The autophagy-lysosome degradation mechanism contributes to the maintenance of cellular homeostasis by removing harmful cytoplasmic materials, such as damaged organelles and unfolded protein aggregates. The accumulation of these materials is among the primary causes of human pathologies, such as neurodegeneration, heart disease, cancer, and infection. Autophagy is initiated by the formation of specialized double-membrane vesicles termed autophagosomes, which sequester and transport such materials to lysosomes for degradation. How autophagy proteins function to generate autophagosomes and how cytotoxic materials are selectively targeted by autophagosomes are unknown. The proposed research seeks to identify key molecular mechanisms underlying autophagosome formation using a combination of biochemical and structural tools. The projects are focused on mechanisms of the autophagy-specific ubiquitin-like proteins (Ubls), Atg8 and Atg12. These Ubls play pivotal roles in autophagosome formation by controlling the unique membrane dynamics. In Aim 1, we will identify and characterize key interactions in the E1-E2-E3 cascade that catalyze the Atg8 lipid conjugation. The E1-E2 and E2-E3 communication will be studied at a biophysical level. Molecular structures containing components of the unique E2-E3 will be determined by X-ray crystallography. In Aim 2, we will determine the membrane and substrate recognition mechanism for Atg8 lipidation. NMR and fluorescence spectroscopy will used to identify structural mechanisms of the recognition. In Aim 3, we will define the structural and biochemical requirements of Atg8-mediated membrane dynamics using a newly developed membrane tethering assay, NMR, and biochemical experiments. The results from these aims will provide mechanistic understanding of the functions of autophagic Ubls and will be a large step toward comprehensive description of autophagosome formation. The impact of the work will be not only on the autophagy field but also on a broad range of biology.